Folate (folic acid) is a vitamin that is essential for the life-sustaining processes of DNA synthesis, replication, and repair. Folate is also important for protein biosynthesis, another process that is central to cell viability. The pteridine compound, methotrexate (MTX), is structurally similar to folate and as a result can bind to the active sites of a number of enzymes that normally use folate as a coenzyme for biosynthesis of purine and pyrimidine nucleotide precursors of DNA and for interconversion of amino acids during protein biosynthesis. Despite its structural similarity to folic acid, methotrexate cannot be used as a cofactor by enzymes that require folate, and instead competes with the folate cofactor for enzyme binding sites, thereby inhibiting protein and DNA biosynthesis and, hence, cell division.
The ability of the folate antagonist methotrexate to inhibit cell division has been exploited in the treatment of a number of diseases and conditions that are characterized by rapid or aberrant cell growth. For example, methotrexate is currently one of the most widely prescribed drugs for the treatment of rheumatoid arthritis, psoriasis, and cancer (Weinblatt et al., Eng. J. Med., 312:818-822 (1985); Kremer and Lee, Arthritis Rheum., 29:822-831 (1986)). Although methotrexate is among the best tolerated of the disease-modifying anti-rheumatic drugs, a major drawback of methotrexate therapy is a troublesome inter-patient variability in the clinical response and an unpredictable appearance of side-effects including gastrointestinal disturbances, alopecia, elevation of liver enzymes, and bone marrow suppression (Weinblatt et al., Arthritis Rheum., 37:1492-1498 (1994); Walker et al., Arthritis Rheum., 36:329-335 (1993)). Several studies in well-controlled clinical trials have demonstrated that methotrexate is effective at decreasing functional disability, with the maximum effect occurring after about six months of therapy. However, recent findings from retrospective studies on a large cohort of patients with rheumatoid arthritis have suggested that methotrexate dosage may be suboptimal in some patients (Ortendahl et al., J. Rheumatol., 29:2084-2091 (2002)). Thus, the lack of efficient therapeutic drug monitoring of methotrexate therapy and difficulty of rapidly individualizing methotrexate dose-maximizing response hampers effective patient treatment.
Methotrexate enters cells through the reduced folate carrier (RFC-1) and is intracellularly activated by folylpolyglutamate synthase to methotrexate polyglutamates (MTXPGs) (Chabner et al., J. Clin. Invest., 76:907-912 (1985)). The γ-linked sequential addition of glutamic acid residues enhances intracellular retention of methotrexate (Allegra et al., Proc. Natl. Acad. Sci. USA, 82:4881-4885 (1985)). The polyglutamation process is in competition with deconjugation by gamma glutamyl hydrolase (GGH) (Rhee et al., Mol. Pharmacol., 53:1040-1046 (1998); Yao et al., Proc. Natl. Acad. Sci. USA, 93:10134-10138 (1996); Panetta et al., Clin. Cancer Res., 8:2423-2429 (2002)), a lysosomal enzyme having high affinity towards long chain polyglutamates. (Masson et al., J. Clin. Invest., 97:73-80 (1996)).
The accumulation of MTXPGs is critical to the pharmacological effects of methotrexate. In vivo, the concentration of MTXPGs in lymphoblasts and erythrocytes appear to correlate with the therapeutic response to methotrexate in patients with leukemia (Dervieux et al., Blood, 100:1240-1247 (2002); Dervieux et al., Arthritis Rheum., in press, (2004)) or rheumatoid arthritis (Angelis-Stoforidis et al., Clin. Exp. Rheumatol., 17:313-320 (1999); Allegra et al., Proc. Natl. Acad. Sci. USA, 82:4881-4885 (1985)). Polyglutamation of methotrexate is thought to promote the sustained inhibition of de novo purine synthesis by 5-aminoimidazole carboxamide-ribonucleotide transformylase (ATIC) (Dervieux et al., Blood, 100:1240-1247 (2002); Allegra et al., supra, (1985)), thereby promoting the build-up of adenosine, a potent anti-inflammatory agent (Baggott et al., Biochem. J., 236:193-200 (1986); Morabito et al., J. Clin. Invest., 101:295-300 (1998); Montesinos et al., Arthritis, 48:240-247 (2003); Cronstein et al., J. Clin. Invest., 92:2675-2682 (1993)). Furthermore, MTXPGs are inhibitors of thymidylate synthase (TS) (Allegra et al., J. Biol. Chem., 260:9720-9726 (1985)). TS methylates deoxyuridine monophosphate to produce deoxythymidylate, providing a unique de novo source of thymidylate.
Part of the large inter-individual variability in the response to methotrexate is related to common polymorphisms in genes implicated in methotrexate pharmacokinetics or pharmacodynamics (Relling and Dervieux, Nat. Rev. Cancer, 1:99-108 (2001)). Recently, a G to A transition in exon 1 (position 80) of RFC-1, resulting in an arginine to histidine substitution at codon 27, was identified (Chango et al., Mol. Genet. Metab., 70:310-315 (2000)). However, the functional consequence of this polymorphism on methotrexate transport has remained unclear (Whetstine et al., Clin. Cancer Res., 7:3416-3422 (2001); Laverdiere et al., Blood, 100:3832-3834 (2002)). Moreover, a recent study of children with acute lymphoblastic leukemia has suggested that the A variant may be associated with poor clinical outcomes as compared with patients having the G/G genotype; individuals carrying the A/A genotype presented higher plasma concentrations of methotrexate compared to those with the G/G or G/A genotypes (Laverdiere et al., supra, (2002)).
Because individual differences in pharmacokinetic and pharmacodynamic parameters can be difficult to predict and because patient genotype affects these parameters, methotrexate treatment can be rendered safer and more effective through patient genotyping. Thus, there exists a need for novel correlations between patient genotypes and efficacy of methotrexate therapy. There also exists a need for new methods of determining or optimizing the efficacy of methotrexate therapy by determining MTXPG levels in a patient through genotyping. The present invention satisfies these needs and provides related advantages as well.